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Anti-Nuclear Antibody
(ANA) Testing
Immunofluorescence (IFA)
versus
Enzyme-Linked Immunosorbent Assay (ELISA)
Systemic Rheumatic Diseases
(SRDs):
• Systemic lupus erythematosus (SLE)
• Rheumatoid arthritis (RA)
• Sjogren’s Syndrome (SS)
• Mixed connective tissue disease (MCTD)
• Scleroderma
• Dermatomyositis/polymyositis (DM/PM)
• Drug-induced lupus
Characteristics:
• Variable age at onset
• Greater frequency in females
• Multi-systems involved
• Symptoms develop gradually
• Extensive overlap in manifestations
Disease complexity makes diagnosis difficult
Diagnosis
• Laboratory testing = important tool
• Specific diagnosis = more appropriate treatment
• Earlier detection/identification = better symptom
management
Best to detect/identify circulating
autoantibodies to the specific cellular antigens
• Three main types
•Immunofluorescence Assay (IFA)
•Enzyme-linked immunosorbent assay (ELISA)
•Multiplex assays
Tests to Detect ANA
• Whole Hep-2 cells are cultured on slides
• Cytoplasm can mask true nuclear reactivity
• Naturally occurring antigens vary in
concentration (SSA low, Scl-70 low, Jo-1 low)
• Antigens in whole cells are soluble, may be
leached off during processing
IFA Test Antigen
• Whole Hep-2 cells are lysed, centrifuged to
concentrate the nuclei
• Other purified antigens are added, boost signal
for all autoantigens (e.g., SSA, Scl-70, Jo-1)
• Coated onto high-affinity binding wells to retain all
signals during processing
Antigen for ELISA over-rides the faults of the IFA
antigen
ELISA Test Antigen
• Individual antigens, cell components are
coated onto multiple beads
• All activities are detected and identified in
parallel
• High cost of test and automation
• Reimbursement for each analyte may be
denied, especially for negative samples (60-80%
of total)
Multiplex Test Antigen
•IFA patterns may suggest but do not identify specific
antibodies
• IFA positive screens must be repeated for titer
• IFA screening/titrating, is labor-intensive
• Microscopic evaluation requires specialized training
Staining patterns/titers will still need to be follow-up
tested to identify specific antibody(ies)
IFA
• ELISA detects the same ANA antibodies as IFA, with
improved sensitivity/specificity
• ELISA returns measurable results in one determination
(no repeating to titrate)
• ELISA is objective, automatable, requires no
specialized training
• ELISA reports out Index Values
ELISA
•ELISA positives can be followup-tested for IFA patterns
and titers, and reported the same as IFA
Staining patterns/titers will still need to be follow-up
tested to identify specific antibody(ies)
ELISA
• Sensitive
• Specific
• Reproducible
• Automatable
• Easy/faster
• Objective
• Index values vary with antibody levels
ELISA Advantages
False Negatives:
• In some SRDs with antibodies to cytoplasmic
constituents
• In Sjogren’s Syndrome,
scleroderma, polymyositis
• If single antibody, at very low level, is present:
- SSA, subacute cutaneous lupus
- dsDNA, ANA negative lupus
IFA Disadvantages
False Positives:
• Known high sensitivity, low specificity
• 33% of normals can be positive
• > 20% healthy relatives
• 75% elderly population
• In other diseases: Chronic Pulmonary Fibrosis-
Chronic Infection- Chronic Hepatitis
IFA Disadvantages
• Labor-intensive, with microscope screening
• Specialized training required
• Microscope optics can vary
• Tests require standardization
• Inconsistencies in preparation of slides can
alter results
• Some laboratories require 2 separate
analysts’ readings
IFA Difficulties
Homogeneous
Antibodies:
- DNP or Histones (drug-induced lupus)
- dsDNA (SLE)
- ssDNA (not disease-specific)
IFA Staining Patterns
Speckled
Antibodies:
- SSA & SSB (SCLE & neonatal SLE)
- Sm (SLE)
- Sm/RNP (SLE, MCTD)
- Scl-70 (Scleroderma)
IFA Staining Patterns
Nucleolar
Antibodies:
- Scl-70 (Scleroderma)
- Nucleolar (Scleroderma)
IFA Staining Patterns
Centromere
Antibodies:
- Centromere
(CREST variant Scleroderma)
IFA Staining Patterns
ANA Screen by Automated
ELISA versus IFA
Time and Labor Cost Analysis
1. ACL, Vol. 19, Number 9, Nov/Dec 2000
A clinical laboratory periodical1 published a
comparative analysis of Manual and Automated
EIA versus IFA testing.
The following summary is based on information
in that comparison.
MANUAL ELISA
Significant time is required to
perform sample dilutions
manually. Incubation times,
washes, reagent addition,
reading, data analysis,
reporting of results require
hands-on technician time.
Total hands-on time:
2.5 hours
AUTOMATED ELISA
Less than 30 minutes
is required for technician to
set reagents and samples
into instrument. The
technician is then free to
perform other laboratory
functions during the assay.
Total hands-on time:
30 minutes
TIME REQUIREMENTS
ANA Screen by Automated
ELISA versus IFA
Time and Labor Cost Analysis
ANA Screen by Automated
ELISA versus IFA
Time and Labor Cost Analysis
1. CAP Workload Recording Method and Personnel Management Manual, CAP, Northfield, IL
1992:III,118.
Cost was estimated for testing 250 samples.
Technician time required for each sample by IFA
was based on the CAP workload Recording
Method and Personnel Management Manual1.
IFA Screen, 11.0 min (2 min. incremental time)
#89554.810. Cost of the tests was not included.
TIME COST1
LABOR COST COMPARISON
ANA Screen by Automated
ELISA versus IFA
Time and Labor Cost Analysis
TIME COST2
Automated ELISA Immunofluorescence (IFA)
60 minute setup $18.00
for assay for
250 samples.
60 minute shut- $18.00
down and reporting.
Total Labor Cost $36.00
8.6 hours, assay $215.00
plus microscope
analysis for
screening
and reporting for
250 samples.
Total Labor Cost $215.00
1. Cost of testing 250 samples by IFA. Estimated hourly rate for trained technologist = $25.00.
2. Cost of testing 250 samples by EIA. Estimated hourly rate for laboratory technician = $18.00.
Dr. Gail Woods
University of Texas Medical Branch
Galveston,TX
ANA TEST STUDY: COMPARISON OF
IFA TO AUTOMATED ELISA
Test Setup:
• N= 510 samples (no diagnosis)
• ANA ELISA Screen (Diamedix Corp) test
MAGO® Plus Automated EIA System
• IFA (Kallestad, Sanofi-Pasteur) at 1:160 as the
initial screening titer, then……
• IFA positives re-tested at titers of 1:160, 1:320, and
1:640. Strong positives were reported as > 1:640.
ANA TEST STUDY
Test Setup:
• Slides independently read by 2 analysts
• Discrepancies between analysts were reviewed
• Results agreed upon after review were recorded
• IFA results were blinded from ELISA results
ANA TEST STUDY:
Results:
• ELISA Index/interpretation were recorded.
• Equivocals (n=33) were excluded.
• ELISA and IFA results agreed for 437/477
(91.6%) samples.
ANA TEST STUDY:
Initial Correlation:
ANA by IFA
POS NEG
POS 114 14
NEG 26 323
ANA ELISA Screen
33 ELISA equivocals were excluded from the calculations
Relative Sensitivity = 114/128 = 89.1 %
Relative Specificity = 323/349 = 92.6 %
Overall Agreement = 437/477 = 91.6 %
ANA TEST STUDY:
Positive/Negative Frequencies:
• 114 / 477 (23.9 %) were positive (+) by IFA and ELISA
• 323 / 477 (67.7 %) were negative (-) by IFA and ELISA
• 40 / 477 (8.4 %) were in disagreement between IFA
and ELISA
ANA TEST STUDY:
By ELISA tests for clinically significant
antibodies:
• anti-dsDNA
• ENA-6 Screen (6 analytes in one well)
• Profile of separate individual ENA assays:
• SSA • SSB • Sm
• Sm/RNP • Scl-70 • Jo-1
Definitions: Subset of ANAs = anti-ENA group of tests = (Extractable
Nuclear Antigen)
•
Discordants were tested:
ANA TEST STUDY:
Eleven of the 26 EIA(+) but IFA(-) samples were positive
or equivocal in one or more of the individual EIA tests.
red = positive yellow = negative in all tests green = equivocal
Interpretation
EIA
Index
ENA-6
Index
SSA
EU/ml
SSB
EU/ml
Sm
EU/ml
Sm/RNP
EU/ml
Scl-70
EU/ml
Jo-1
EU/ml
dsDNA
IU/ml
Pos > 1.1 > 1.1 > 20 > 20 > 20 > 20 > 20 > 20 > 35
Neg <0.9 < 0.9 < 16 < 16 < 16 < 16 < 16 < 16 < 25
1 9.72 1.369 5.8 0.7 4.3 6.4 3 7.6 25.5
2 4.15 0.318 3.3 2.2 1.4 2.1 1.7 1.8 127.6
3 4.09 0.554 4.9 2.8 2.1 4 2.9 2.7 154.0
4 4.01 0.792 7.2 3.4 3.2 6.2 3.7 4.1 212.7
5 3.57 7.831 105.8 0.8 0.8 1.1 1 0.6 13.1
6 3.12 3.531 68.3 2.2 2.2 5.1 1.9 2.4 33.7
7 3.11 0.316 2.9 1.7 2.4 2.4 2.5 2.3 31.7
8 1.95 1.267 4.3 25.9 1.5 5.1 1.9 2.1 10.4
9 1.27 0.294 1.4 0.5 2.2 3.4 1.2 3.5 31.3
10 1.26 0.298 3.1 0.7 1.1 3.2 1.6 1.7 35.6
11 1.16 0.339 4.6 1.5 2.3 4.2 3.3 2.8 47.2
26 Samples POS by EIA / NEG by IFA
ANA TEST STUDY:
The remaining twelve of the EIA(+) IFA(-) samples were negative
in the individual EIA tests.* 3 samples were unavailable for follow-up testing
red = positive
Interpretation
EIA
Index
ENA-6
Index
SSA
EU/ml
SSB
EU/ml
Sm
EU/ml
Sm/RNP
EU/ml
Scl-70
EU/ml
Jo-1
EU/ml
dsDNA
IU/ml
Pos > 1.1 > 1.1 > 20 > 20 > 20 > 20 > 20 > 20 > 35
Neg <0.9 < 0.9 < 16 < 16 < 16 < 16 < 16 < 16 < 25
1 6.57 0.394 4.0 2.4 2.7 2.1 1.8 3.3 9.6
2 5.88 0.673 5.7 0.9 1.2 3.2 1.3 1.8 9.4
3 1.84 0.614 11.7 13.8 11.8 8.7 12.5 10 14.2
4 1.84 0.214 4.0 0.6 0.8 1.6 1.2 1.5 6.4
5 1.60 0.216 2.0 0.9 1 1.5 1.5 1.5 8.0
6 1.59 0.256 2.1 0.4 1 1.5 0.8 1.3 17.1
7 1.56 0.478 3.7 0.4 0.9 2 1.6 1.9 7.1
8 1.52 0.302 2.2 0.3 0.5 1.7 1.5 0.6 7.2
9 1.45 0.397 5.6 1.6 2.5 4 2.9 2.6 13.2
10 1.35 0.211 3.8 1.9 2.1 2.7 2.4 1.9 19.8
11 1.28 0.279 2.0 0.4 0.6 5.3 0.9 1.5 5.2
12 1.18 0.434 3.4 1.2 2 2.8 3 3.1 13.4
26 Samples POS by EIA / NEG by IFA
ANA TEST STUDY:
• 8 / 23 ELISA (+) and IFA (-) samples were positive and
3 were equivocal in one or more of the profile of tests for
antibodies of clinical significance (e.g., SSA or SSB or dsDNA).
• Significant number “false negative” by IFA ?
• ANA by IFA has been reported not to detect antibodies
such as SSA (subacute cutaneous lupus) and dsDNA (ANA negative
lupus), especially when they occur alone, in the absence of
other antibodies.
Results:
ANA TEST STUDY:
red=positive yellow = negative in all tests green=equivocalP
t
. IFA Titer
EIA
Index
ENA-6
Index
SSA
EU/ml
SSB
EU/ml
Sm
EU/ml
Sm/RNP
EU/ml
Scl-70
EU/ml
Jo-1
EU/ml
Pos > 1.1 > 1.1 > 20 > 20 > 20 > 20 > 20 > 20
Neg <0.9 < 0.9 < 16 < 16 < 16 < 16 < 16 < 16
160 0.88 0.274 1.4 1.1 0.6 3.9 1.3 1.4
1280 0.85 0.147 4.8 0.7 1.0 1.7 1.4 1.7
640 0.85 0.202 4.0 0.6 1.0 1.6 1.1 2
320 0.85 0.223 1.9 0.2 0.7 1.8 1.4 1.1
160 0.85 0.316 1.5 0.5 1.1 4.0 1.5 1.8
640 0.85 0.188 2.4 0.3 0.8 1.2 0.7 0.8
320 0.85 1.855 18.2 1.3 1.4 2.0 1.7 2.3
160 0.85 0.493 2.9 1.2 3.8 5.2 2.8 4.4
160 0.85 0.283 2.6 1.6 1.0 1.2 3.0 0.9
160 0.85 0.149 2.9 1.2 0.9 2.0 1.2 0.9
160 0.85 0.131 2.6 0.6 0.9 1.3 1.3 1.4
320 0.85 0.191 4.7 1.5 1.1 2.1 0.9 1.3
320 0.85 0.302 2.4 0.3 1.3 1.9 1.5 1.1
160 0.85 0.204 2.4 2.5 1.0 1.3 1.2 1.2
14 Samples NEG by EIA / POS by IFA
Thirteen of the 14 EIA (-) but IFA (+) samples
were negative in the individual EIA tests.
ANA TEST STUDY:
• 13 of the 14 samples ELISA (-) and IFA (+) were
negative by the profile of individual assays
• The 1 remaining sample was equivocal in the SSA
test and positive in the ENA-6 Screen (as a result
of the low level of anti-SSA)
• Majority “false positive” by IFA?
Results:
ANA TEST STUDY:
Total Discordant Resolution:
• 13 of the 14 samples IFA(+) / ELISA (-) were negative
by the profile tests, in agreement with ELISA
• 8 of the 26 samples IFA(-) / ELISA (+) were positive (and
3 were equivocal) by the profile tests, in agreement with
ELISA
• 3 Samples IFA(-) / ELISA (+) were QNS for discordant
testing
ANA TEST STUDY:
Correlation Retabulated :
36 Equivocals were excluded from the calculations
ANA by IFA
POS NEG
POS 122 1
NEG 12 336
ANA ELISA Screen
Relative Sensitivity = 122/122 = 100.0 %122/123 = 99.20%
Relative Specificity = 336/378 = 96.6 %
Overall Agreement = 458/470 = 97.4 % 458/471 = 97.20%
ANA by IFA
POS NEG
POS 114 14
NEG 26 323
ANA ELISA Screen
Initial Results
Relative Sensitivity = 114/128 = 89.1 %
Relative Specificity = 323/349 = 92.6 %
Overall Agreement = 437/477 = 91.6 %
33 Equivocals were excluded
ANA TEST STUDY:
What to do with Equivocals?
• In this study, 28/33 (84.8%) equivocals were
negative by IFA
• 3 were positive with a titer of 1:160, 1 with a titer
of 1:640, 1 was not titered.
• Suggestions (per Diamedix package insert):
(1) Report out as ‘equivocal’, or retest; if positive retest,
report as ‘positive’; if negative retest, report as ‘negative’.
(2) Test by another test
(3) Test a new sample
ANA TEST STUDY:
• To ‘rule out’ negatives:
Approx. 60-80% of the total population tested
• To reduce IFA false positive and negatives
• Positive samples can be tested by IFA for staining
pattern and titer
• ELISA Index varies with IFA titer
Benefits of the ANA ELISA Screen
ANA TEST STUDY:
Mean = 2.14 Mean = 2.02 Mean = 5.54 Mean = 7.66
Median = 1.22 Median = 1.83 Median = 4.06 Median = 8.79
High = 12.60 High = 5.62 High = 12.60 High = 12.60
Low = 0.39 Low = 0.40 Low = 0.68 Low = 0.85
160 Titer (n=27) 320 Titer (n=28) 640 Titer (n=49) 1280 Titer (n=27)
ANA ELISA Screen Index vs IFA Titer
0
2
4
6
8
10
12
14
0 200 400 600 800 1000 1200 1400
IFA Titer
AN
A E
LIS
A S
cre
en
In
de
x
ANA TEST STUDY:
Although the IFA test at each titer does not reflect the
wide positive response of the ELISA test, there is
general correlation between IFA titer and ELISA Index:
• 1:160 versus 1.22 median index
• 1:320 versus 1.83 median index
• 1:640 versus 4.06 median index
• 1:1280 versus 8.79 median index
ANA TEST STUDY:
IFA
• Results subjective (analyst
interpretation)
• Multiple dilutions and repeat
testing required
• 13/14 discordants (+) by IFA
were negative in profile tests
• 11/23 (47.8%) discordants (-) by
IFA were positive/equivocal in
profile tests *
ELISA
• Results objective
(spectrophotometric)
• Index values obtained from
a single well
• 13/14 discordants (-) by ELISA
were negative in profile tests
(1 SSA equivocal)
• 11/23 (47.8%) discordants
(+) by ELISA were positive/
equivocal in profile tests *
Conclusions:
*11 samples were positive/equivocal for antibody to dsDNA and/or SSA and SSB
References
Homburger, H A et al. 1998. Detection of Antinuclear Antibodies. Comparative Evaluation of Enzyme Immunoassay and Indirect Immunofluorescence Methods. Arch Pathol Lab Med Vol 122:993-999.
Jacob GL. 2001. Clinical Laboratory Experience with Enzyme Immunoassays for Auto-Antibodies to Nuclear Antigens. AMLI Presentation. San Diego, Aug 2001.
Egner, W. 2000. The use of laboratory tests in the diagnosis of SLE. J Clin Pathol. 53:424-432.
Photo Credits: IFA images courtesy of University of Washington
